This invention relates to analogs of primary amide bonds and methods of using such analogs such as for eliciting catalytic antibodies for hydrolysis of primary amide bonds. The invention also relates to catalytic antibodies capable of catalyzing the hydrolysis or formation of primary amide bonds.
This invention further relates to novel methods for using such catalytic antibodies as therapeutic agents wherein said catalytic antibodies cause hydrolysis of primary amide bonds of peptides and proteins.
This invention also relates to novel methods for using such catalytic antibodies as catalysts for hydrolysis of formation of primary amide bonds in the synthesis of chemicals.
Still further, this application relates to novel methods for selecting or screening for catalytic activity by catalytic antibodies in samples containing antibodies as well as other biological molecules. Such methods for selecting or screening are rapid and efficient and thus, allow the routine screening of larger numbers of potential catalytic antibodies.
Various documents are cited parenthetically throughout the text of this disclosure, with full citation to these documents appearing as a list immediately preceding the claims. Other documents are cited parenthetically, in full, throughout the text of this disclosure. These documents pertain to the field of this invention, and each of these documents is hereby incorporated herein by reference.
One of the most frequently cited goals of catalytic antibody research is the generation of antibodies capable of efficient, specific hydrolysis of amide bonds (1-14). This goal partly results from the scientific challenge of generating an antibody to catalyze a relatively kinetically inert reaction and partly from the potentially vast range of practical applications for proteolytic antibodies.
Until now, no haptens have been designed which have demonstrably elicited antibodies that hydrolyze unactivated amides without cofactor assistance.
The difficulty in eliciting amide-hydrolyzing antibodies stems primarily from the difficulties associated with screening the antibodies for catalysis. The uncatalyzed rate (kuncat) of amide hydrolysis (with a half-life on the order of 7 years at neutral pH (15)) is much slower than that of typical esters (16), carbonates (17), or activated amides (8, 18). Correspondingly, it is much more difficult to detect antibodies that catalyze amide hydrolysis. If two separate reactions of different uncatalyzed rates (kuncat) are catalyzed by two antibodies with identical rate enhancements (kcat/kuncat), it will be more difficult to detect the antibody that is slower in absolute terms (kcat). There are two main reasons for this. One reason is that in the slower reaction there will be fewer turnovers per unit of time and the assay system must be able to detect extremely low levels of formed product. The second reason is that reactions with slower kuncat values typically require longer periods of antibody-substrate incubation (days or weeks) than do faster reactions (minutes or hours). Long incubation periods may lead to false results because of the possible appearance of by-products, the antibody may denature, especially at non-neutral pH values, and traces of adventitious enzymes may catalyze the reaction in question.
As mentioned above, until now no catalytic antibodies have been generated that are capable of unassisted catalytic hydrolysis of an unactivated amide bond. Documentation of rationally-designed catalytic antibody transformations of amide bonds have been limited to antibodies capable of activated amide hydrolysis (8), metallo-cofactor-assisted amide hydrolysis (20), and amide bond rearrangement (21). Also, naturally-occurring peptide hydrolysis by autoantibodies have been reported (22). It should be pointed out that in the last example the reported antibody activities were discovered rather than designed. Here we describe the first successful design of a catalytic antibody capable of unassisted hydrolysis of an unactivated amide bond.
In nature, the most commonly encountered amide bonds are found in peptides and proteins. Three principle types of amide bonds are found in peptide and proteins: as peptide bonds linking individual amino acids, in asparagine and glutamine amino acid side chains, and at the C-terminus of some peptides (i.e. C-terminal carboxamide). Hydrolyses of both types of amide bonds, peptide bonds and primary amide bonds (asparagine, glutamine and C-terminal amides), have the same or very similar Gibbs free energies of activation (23-25). Hence, they require the same amount of transition state stabilization to be catalyzed with similar rate enhancements.
Amidated peptide hormones that are important in disease states include: calcitonin (29), calcitonin gene-related peptide (30,31), big gastrin (32,33), and bombesin-like peptides [BLP, otherwise known as bombesin-related peptides (BRP) or gastrin-releasing peptides (GRP)] (34,35). Elevated calcitonin levels have been associated with several disease states including medullary thyroid carcinoma, C-cell hyperplasia, chronic renal failure, pancreatitis, mineral and bone disorders, and hyperthyroidism (29). Elevated gastrin is associated with gastrinomas, BLP with small cell lung cancer (SCLC) (33). Experimental treatments are underway to neutralize the effects of these hormones in disease states using monoclonal antibodies (anti-BLP)(36), neutral endopeptidase 24.11 (BLP hydrolysis)(37), and synthetic antagonists (BLP)(38-41).
Also of potential therapeutic relevance are peptides and proteins containing amino acids with amide side chains (e.g., asparagine and glutamine). Administration of bacterial asparaginase, which hydrolyzes the side chain carboxamide of free asparagine, has been shown to be an effective anti-leukemia agent (42).
U.S. patent application Ser. No. 190,271 of which this application is a continuation-in-part, describes analogs of peptide bonds and methods for eliciting catalytic antibodies which hydrolyze such peptide bonds. The analogs and antibodies described therein are directed towards the modification of peptide bonds which are secondary amide bonds whereas the present application is related to modification of primary amide bonds.
U.S. patent application Ser. No. 08/007,684 which is a continuation-in-part of International Application WO Serial No. 92/01047 (PCT/GB91/01134) describes the isolation and production of catalytic antibodies displayed on bacteriphage and the isolation and production of human catalytic antibodies. The disclosures of both of these applications are hereby incorporated herein by reference.
Based on the observations described above, it is desired to develop transition state analogs of primary amides to elicit catalytic antibodies which can cleave primary amide bonds in peptides and proteins.
It is desired to employ catalytic antibodies which catalyze the hydrolysis or formation of primary amide bonds as therapeutic agents by administration to living organisms. Modification of primary amide bonds in target peptides and proteins would expectedly alter their physiological function and therefore provide beneficial therapeutic effect on the living organisms.
The invention herein described provides methods for the production of catalytic antibodies which can hydrolyze primary amide bonds. Such antibodies can be directed to hydrolyze and/or form primary amide bonds specifically in selected peptides and other molecules. A direct result of this invention is the production of many different therapeutic products having benefit to the public which could not be developed based on the prior art.
It has surprisingly been found that compounds of this invention elicit antibodies which enhance the rate of hydrolysis (cleavage) or formation of primary amide bonds and, that such catalytic antibodies have therapeutic and industrial utility.
The present invention accordingly provides a compound of formula (I): 
wherein:
Y is a polypeptide,
R1 is bonded to the N-terminus of Y and is hydrogen or a branched or linear, substituted or unsubstituted, C1-21 alkyl, alkene, or alkyne group,
R2 is a side chain of a naturally occuring amino acid,
and X is 
The present invention further provides for a compound of formula (I) wherein the polypeptide, Y, consists of the polypeptide sequence of a protein containing a C-terminal carboxamide with the C-terminal amino acid is omitted.
Also contemplated by the present invention are compounds of formula (i) wherein R1 comprises a reactive group such as 
where n is an integer of 1 to 21, attached to the N-terminal amino acid of the polypeptide such that the comound of formula (I) can be chemically reacted with and attached to a carrier molecule.
Accordingly, immunogens are contemplated by the present invention wherein the compounds of formula (I) are attached to a carrier molecule.
The present invention also provides a compound of formula (II):
Rxe2x80x94Yxe2x80x94[(CH2)nxe2x80x94X]mxe2x80x83xe2x80x83(II)
where in:
R is hydrogen or a branched or linear, substituted or unsubstituted, C1-21 alkyl, alkene, or alkyne group,
Y is a polypeptide,
the xe2x80x94(CH2)nxe2x80x94X groups replace side chains of asparagine or glutamine of said polypeptide,
n is 1 or 2,
m is an integer greater than or equal to one and less than or equal to the total number of asparagine and glutamine amino acids in said polypeptide,
and X is 
Also contemplated by the present invention are compounds of formula (ii) wherein R1 comprises a reactive group such as 
where n is an integer of 1 to 21, attached to the polypeptide such that the compound of formula (ii) can be chemically reacted with and attached to a carrier molecule.
Accordingly, immunogens are contemplated by the present invention wherein the compounds of formula (ii) are attached to a carrier molecule.
The present invention also provides for compounds of the formula (I) or (II) in which the peptide consists of 1 to 20 naturally occuring amino acids.
The present invention further provides a compound of the formula (III): 
wherein:
R1 is a branched or linear, substituted or unsubstituted, C1-21 alkyl, alkene, or alkyne group,
R2 is a side chain of a naturally occuring amino acid,
and X is 
Also contemplated by the present invention are compounds of formula (III) wherein R1 comprises a reactive moiety such as 
where n is an integer of 1 to 21, such that the comound of formula (III) can be chemically reacted with and attached to a carrier molecule.
Accordingly, immunogens are contemplated by the present invention wherein the compounds of formula (III) are attached to carrier molecules.
The present invention further contemplates antibodies which are capable of enhancing the rate of hydrolysis and/or the rate of formation of a primary amide bond.
Also contemplated are antibodies which are capable of enhancing the rate of hydrolysis and/or the rate of formation of a primary amide bond produced by the method comprising the steps of:
(i) generating a gene library of antibody-derived domains;
(ii) inserting coding for said domains into a phage expression vector; and
(iii) isolating said catalytic antibodies.
Further contemplated are antibodies which are capable of enhancing the rate of hydrolysis and/or the rate of formation of a primary amide bond produced by the method comprising the steps of:
(i) immunizing an animal with a composition comprising an analog of a reactant, reaction intermediate, or product of the hydrolysis reaction;
(ii) removing antibody-producing lymphocytes from said animal; and,
(iii) fusing the lymphocytes with myeloma cells and thereby producing hybridoma cells producing the antibody.
Also contemplated are antibodies which are capable of enhancing the rate of hydrolysis and/or the rate of formation of a primary amide bond obtained by the method comprising:
(i) raising antibodies against a hapten
(ii) immobilizing said antibodies
(iii) adding a substrate to said antibodies, and
(iv) identifying antibodies capable of catalyzing the conversion of said substrate to a product.
Further contemplated are antibodies obtained by the above method further comprising, after step (i):
(i) concentrating said antibodies and
(ii) purifying said antibodies.
Also provided by the present invention are methods for providing therapy in an animal or individual in need of said therapy comprising administering to said individual or animal an antibody capable of enhancing the rate of hydrolysis and/or formation of a primary amide bond. The animal or individual can be a patient.